1. Field of the Invention
The present application relates generally to microvesicles and their isolation and analysis, including methods of identifying populations of invasive microvesicles that contain the protein ARF6. As described in the examples below, invasive microvesicles from tumor cells contain a variety of specific proteins, including ARF6.
2. Description of the Related Art
Microvesicles are small, membrane-enclosed structures that are shed from a variety of cell types and can contain a variety of bioactive molecules, including nucleic acids and proteins. Microvesicle shedding by the outward fission of membrane vesicles from the cell surface is a selective process that occurs more frequently in certain cells, such as cancerous tumor cells. These released microvesicles (also referred to as microparticles, particles, and ectosomes) have been widely detected in various biological fluids, including peripheral blood, urine, saliva, and ascites.
Microvesicles are believed to facilitate various processes, including tumor invasion and metastasis, and are also believed to play a role in inflammation, coagulation, stem-cell renewal and expansion, evasion of the immune response, and bone mineralization. The composition of microvesicles, and thus their function, varies depending on the cells from which they originate. For example, microvesicles secreted by skeletal cells have been found to play a role in initiating bone mineralization, while microvesicles secreted from endothelial cells have been implicated in angiogenesis. Microvesicles are thought to play a role in metastasis by facilitating angiogenesis, escape from immune surveillance, and extra-cellular matrix (ECM) degradation. Proteolytic activity associated with microvesicles shed by tumor cells has been found to correlate with disease stage.
The process of metastasis occurs when cells detach from a primary tumor and invade surrounding tissues to reach other, distal locations. This process leads to the formation of secondary tumors, and is one of the life-threatening hallmarks of malignant cancer. Standardized screening methods and techniques that are sensitive enough to detect early-stage cancer and other diseases are currently unavailable for a variety of conditions, including ovarian, prostate, breast, glioma, and melanoma cancers. As a result, metastasis often occurs before a patient can be diagnosed and treated.
Recent studies show that a variety of molecules are involved with the complex process of metastasis. One such molecule is the protein known as ARF6, of the ARF family of small GTP-binding proteins, which regulates membrane trafficking and actin cytoskeleton remodeling and has a role in acquisition of migratory and invasive potential of cancer and other cell types. Recent studies utilizing in vitro cell invasion assays have indicated that in invasive melanoma, glioma, and breast cancer cell lines, the ARF6 GTP/GDP cycle can regulate the invasive potential of the cells. In addition, cellular depletion of ARF6 by siRNA or inhibition of ARF6 activation by expression of a dominant negative ARF6 mutant attenuates tumor cell invasion in vitro. Recent animal studies have also revealed a role for ARF6 activation in melanoma and glioma cell invasion (V. Muralidharan-Chari et al., Cancer Res. 69, 2201-09 (2009), B. Hu et al., Cancer Res. 69, 794-801 (2009)). Moreover, screening of various breast tumor cell lines reveals a direct correlation between ARF6 protein expression and invasive capacity (S. Hashimoto et al., Proc. Natl. Acad. Sci. 101, 6647-52 (2004)). In addition, a molecule known as ARF6 exchange factor GEP 100 is expressed in 70% of primary breast ductal carcinomas, and is preferentially co-expressed with EGFR in malignant tumors (M. Morishige et al., Nat. Cell Biol. 10, 85-92 (2008)).